Protein transduction domains (PTDs), also known as cell penetrating peptides (CPPs), are oligo- or poly-cationic peptides that can facilitate cellular uptake of many different cargos such as small molecules, proteins, DNA/RNA and nanoparticles.
In 1988, Frankel and Pabo, and Green and Lowestein independently reported that TAT protein from HIV is able to cross cellular membranes and localize inside cells. (Frankel, et al. 1988 Cell 55, 1189-1193; Green, et al. 1988 Cell 55, 1179-1188.) Since then, protein transduction domains have been under intense study for two major reasons. First, it is well known that the plasma membrane limits the transport of highly charged molecules. The fact that PTDs, with multiple cationic centers, readily transverse the membrane is important for a fundamental understanding of membrane transport. Second, the ability of PTDs to deliver cargo (proteins, antibodies, and nucleic acids) into mammalian cells offers possibilities for both new therapies and new tools to study cell biology. (Fonseca, et al. 2009 Adv. Drug. Deliv. Rev. 61, 953-964; Gump, et al. 2007 TRENDS Mol Med 13, 443-448; Sebbage, 2009 Bioscience Horizons 2, 64-72.)
PTDs primarily consist of cationic amino acid sequences such as arginines and/or lysines. Early studies showed that the translocation abilities of PTDs were directly associated with the presence of arginine residues. (Schwarze, et al. 2000 Trends Pharmacol Sci 21, 45-48; Futaki, et al. 2003 J. Mol. Recog. 16, 260-264; Fischer, et al. 2000 J. Peptide Res. 55, 163-172; Mitchell, et al. 2000 J. Peptide Res. 56, 318-325; Futaki, et al. 2001 J. Biol. Chem. 276, 5836-5840; Wender, et al. 2000 Proc. Natl. Acad. Sci. USA 97, 13003-13008.) For example, in the case of TAT49-57 (RKKRRQRRR), replacement of the arginine amino acids with alanine or other cationic residues (lysine, histidine, and orthonine), led to reduced cellular uptake. In contrast, substitution of all non-arginine residues with arginine (i.e. Arg-replacement) resulted in enhanced internalization efficiency (e.g., R9 was reported to be 20-fold more efficient than TAT49-57). In addition to arginine content, the peptide length sets another parameter for cellular uptake. It was reported that there is an optimum length for maximum activity. (Rothbard, et al. 2002 J. Med. Chem. 45, 3612-3618.)
Although the number of known PTDs has increased significantly and small molecule synthetic analogues have been attempted, design and synthesis of simple structures that capture the biological activity of peptides, proteins, and oligonucleotides remains an important challenge. (e.g., Lienkamp, et al. 2008 J. A. Chem. Soc. 130, 9836-9843; Gabriel, et al. 2008 Biomacromolecules 9, 2980-2983.) There is a significant unmet need for novel approaches, compositions and methods that provide synthetic mimics of PTDs having improved cell-penetrating properties.